This invention relates to pharmaceutically active compounds which inhibit the vitronectin receptor and are useful for the treatment of inflammation, cancer and cardiovascular disorders, such as atherosclerosis and restenosis, and diseases wherein bone resorption is a factor, such as osteoporosis.
Integrins are a superfamily of cell adhesion receptors, which are transmembrane glycoproteins expressed on a variety of ceils. These cell surface adhesion receptors include gpIIb/IIIa (the fibrinogen receptor) and xcex1vxcex23 (the vitronectin receptor). The fibrinogen receptor gpIIb/IIIa is expressed on the platelet surface, and mediates platelet aggregation and the formation of a hemostatic clot at the site of a bleeding wound. Philips, et at., Blood., 1988, 71, 831. The vitronectin receptor xcex1vxcex23 is expressed on a number of cells, including endothelial, smooth muscle, osteoclast, and tumor cells, and, thus, it has a variety of functions. The xcex1vxcex23 receptor expressed on the membrane of osteoclast cells mediates the adhesion of osteoclasts to the bone matrix, a key step in the bone resorption process. Ross, et al., J. Biol. Chem., 1987, 262, 7703. A disease characterized by excessive bone resorption is osteoporosis. The xcex1vxcex23 receptor expressed on human aortic smooth muscle cells mediates their migration into neointima, a process which can lead to restenosis after percutaneous coronary angioplasty. Brown, et al., Cardiovascular Res., 1994, 28, 1815. Additionally, Brooks, et al., Cell, 1994, 79, 1157 has shown that an xcex1vxcex23 antagonist is able to promote tumor regression by inducing apoptosis of angiogenic blood vessels. Thus, agents that block the vitronectin receptor would be useful in treating diseases, such as osteoporosis, restenosis and cancer.
The vitronectin receptor is now known to refer to three different integrins, designated xcex1vxcex21, xcex1vxcex23 and xcex1vxcex25. Horton, et al., Int. J. Exp. Pathol, 1990, 71, 741. xcex1vxcex21 binds fibronectin and vitronectin. xcex1vxcex23 binds a large variety of ligands, including fibrin, fibrinogen, laminin, thrombospondin, vitronectin, von Willebrand""s factor, osteopontin and bone sialoprotein I. xcex1vxcex25 binds vitronectin. The vitronectin receptor xcex1vxcex25 has been shown to be involved in cell adhesion of a variety of cell types, including microvascular endothelial cells, (Davis, et al., J. Cell. Biol., 1993, 51, 206), and its role in angiogenesis has been confirmed. Brooks, et al., Science, 1994, 264, 569. This integrin is expressed on blood vessels in human wound granulation tissue, but not in normal skin.
The vitronectin receptor is known to bind to bone matrix proteins which contain the tri-peptide Arg-Gly-Asp (or RGD) motif. Thus, Horton, et al. Exp. Cell Res. 1991, 195. 368. disclose that RGD-containing peptides and an anti-vitronectin receptor antibody (23C6) inhibit dentine resorption and cell spreading by osteoclasts In addition. Sato, et al. J. Cell Biol. 1990, 111, 1713 discloses that echistatin, a snake venom peptide which contains the RGD sequence, is a potent inhibitor of bone resorption in tissue culture and inhibits attachment of osteoclasts to bone.
It has now been discovered that certain compounds are potent inhibitors of the xcex1vxcex23 and xcex1vxcex25 receptors. In particular, it has been discovered that such compounds are more potent inhibitors of the vitronectin receptor than the fibrinogen receptor
This invention comprises compounds of the formula (I) as described hereinafter, which have pharmacological activity for the inhibition of the vitronection receptor and are useful in the treatment of inflammation, cancer and cardiovascular disorders, such as atherosclerosis and restenosis, and diseases wherein bone resorption is a factor, such as osteoporosis.
This invention is also a pharmaceutical composition comprising a compound according to formula (I) and a pharmaceutically carrier.
This invention is also a method of treating diseases which are mediated by the vitronectin receptor. In a particular aspect, the compounds of this invention are useful for treating atherosclerosis, restenosis, inflammation, cancer and diseases wherein bone resorption is a factor, such as osteoporosis.
This invention comprises novel compounds which are more potent inhibitors of the vitronectin receptor than the fibrinogen receptor. The novel compounds comprise a dibenzocycloheptene core in which a nitrogen-containing substituent is present on one of the aromatic six-membered rings of the dibenzocycloheptene and an aliphatic substituent containing an acidic moiety is present on the seven-membered ring of the dibenzocycloheptene. The dibenzocycloheptene ring system is believed to orient the substituent sidechains on the six and seven membered rings so that they may interact favorably with the vitronectin receptor. It is preferred that about twelve to fourteen intervening covalent bonds via the shortest intramolecular path will exist between the acidic group on the aliphatic substituent of the seven-membered ring of the dibenzocycloheptene and the nitrogen of the nitrogen-containing substituent on one of the aromatic six-membered ring of the dibenzocycloheptene.
This invention comprises compounds of formula (I): 
or a pharmaceutically acceptable salt thereof.
Also included in this invention are pharmaceutically acceptable addition salts and complexes of the compounds of this invention. In cases wherein the compounds of this invention may have one or more chiral centers, unless specified, this invention includes each unique nonracemic compound which may be synthesized and resolved by conventional techniques. In cases in which compounds have unsaturated carbonxe2x80x94carbon double bonds, both the cis (Z) and trans (E) isomers are within the scope of this invention. In cases wherein compounds may exist in tautomeric forms, such as keto-enol tautomers, such as 
and each tautomeric form is contemplated as being included within this invention whether existing in equilibrium or locked in one form by appropriate substitution with Rxe2x80x2.
The compounds of formula (I) inhibit the binding of vitronectin and other RGD-containing peptides to the vitronectin receptor. Inhibition of the vitronectin receptor on osteoclasts inhibits osteoclastic bone resorption and, is useful in the treatment of diseases wherein bone resorption is associated with pathology, such as osteoporosis and osteoarthritis.
In another aspect, this invention is a method for stimulating bone formation which comprises administering a compound which causes an increase in osteocalcin release. Increased bone production is a clear benefit in disease states wherein there is a deficiency of mineralized bone mass or remodeling of bone is desired, such as fracture healing and the prevention of bone fractures. Diseases and metabolic disorders which result in loss of bone structure would also benefit from such treatment. For instance, hyperparathyroidism, Paget""s disease, hypercalcemia of malignancy, osteolytic lesions produced by bone metastasis, bone loss due to immobilization or sex hormone deficiency, Behcet""s disease, osteomalacia, hyperostosis and osteopetrosis, could benefit from administering a compound of this invention.
Additionally, since the compounds of the instant invention inhibit vitronectin receptors on a number of different types of cells, said compounds would be useful in the treatment of inflammatory disorders, such as rheumatoid arthritis and psoriasis, and cardiovascular diseases, such as atherosclerosis and restenosis. The compounds of Formula (I) of the present invention may be useful for the treatment or prevention of other diseases including, but not limited to, thromboembolic disorders, asthma, allergies, adult respiratory distress syndrome, graft versus host disease, organ transplant rejection, septic shock, eczema, contact dermatitis, inflammatory bowel disease, and other autoimmune diseases. The compounds of the present invention may also be useful for wound healing.
The compounds of the present invention are also useful for the treatment, including prevention, of angiogenic disorders. The term angiogenic disorders as used herein includes conditions involving abnormal neovascularization. Where the growth of new blood vessels is the cause of, or contributes to, the pathoogy associated with a disease, inhibition of angiogenisis will reduce the deleterious effects of the disease. An example of such a disease target is diabetic retinopathy. Where the growth of new blood vessels is required to support growth of a deleterious tissue, inhibition of angiogenisis will reduce the blood supply to the tissue and thereby contribute to reduction in tissue mass based on blood supply requirements. Examples include growth of tumors where neovascularization is a continual requirement in order that the tumor grow and the establishment of solid tumor metastases. Thus the compounds of the present invention inhibit tumor tissue angiogenesis, thereby preventing tumor metastasis and tumor growth.
Thus, according to the methods of the present invention, the inhibition of angiogenesis using the compounds of the present invention can ameliorate the symptoms of the disease, and, in some cases, can cure the disease.
Another therapeutic target for the compounds of the instant invention are eye diseases chacterized by neovascuiarization. Such eye diseases include corneal neovascular disorders, such as corneal transplantation herpetic keratitis, luetic keratitis, pterygium and neovascular pannus associated with contact lens use. Additional eye diseases also include age-related macular degeneration, presumed ocular histoplasmosis, retinopathy of prematurity and neovascular glaucoma.
This invention further provides a method of inhibiting tumor growth which comprises administering stepwise or in physical combination a compound of formula (I) and an antineoplastic agent, such as topotecan and cisplatin.
With respect to formula (I): 
Representative of the novel compounds of this invention are the following:
(xc2x1)-10,11-Dihydro-3-[2-(6-aminopyridin-2-yl)-1-ethoxy]-5H-dibenzo[a,d]cycloheptene-10-acetic acid;
(xc2x1)-10,11-Dihydro-3-[4-(pyridin-2-ylamino)-1-butyl]-5H-dibenzo[a,d]cycloheptene-10-acetic acid;
(xc2x1)-10,11-Dihydro-3-[3-(4-ethoxypyridin-2-ylamino)-1-propyloxy]-5H-dibenzo[a,d]cycloheptene-10-acetic acid;
(S)-10,11-Dihydro-3-[3-(pyridin-2-ylamino)-1-propyloxy]-5H-dibenzo[a,d]cycloheptene-10-acetic acid;
(R)-10,11-Dihydro-3-[3-(pyridin-2-ylamino)-1-propyloxy]-5H-dibenzo[a,d]cycloheptene-10-acetic acid;
(xc2x1)-10,11-Dihydro-3-[3-(3,4,5,6-tetrahydropyrimidin-2-ylamino)-1-propyloxy]-5H-dibenzo[a,d]cycloheptene-10-acetic acid;
(xc2x1)-10,11-Dihydro-3-[2-[2-(ethylamino)thiazol-4-yl]-1-ethoxy]-5H-dibenzo[a,d]cycloheptene-10-acetic acid;
(xc2x1)-10,11-Dihydro-3-[3-(isoquinoline-1-ylamino)-1-propyloxy]-5H-dibenzo[a,d]cycloheptene-10-acetic acid;
(xc2x1)-10,11-Dihydro-7-fluoro-3-[3-(pyridin-2-ylamino)-1-propyloxy]-5H-dibenzo[a,d]cycloheptene-10-acetic acid;
(S)-10,11-Dihydro-3-[3-(4-methylpyridin-2-ylamino)-1-propyloxy]-5H-dibenzo[a,d]cycloheptene-10-acetic acid;
(S)-10,11-Dihydro-3-[3-(4-ethoxypyridin-2-ylamino)-1-propyloxy]-5H-dibenzo[a,d]cycloheptene-10-acetic acid;
(xc2x1)-10,11-Dihydro-6-methyl-3-[3-(pyridin-2-ylamino)-1-propyloxy]-5H-dibenzo[a,d]cycloheptene-10-acetic acid;
(xc2x1)-10,11-Dihydro-2-(dimethylamino)methyl-7-fluoro-3-[3-(pyridin-2-ylamino)-1-propyloxy]-5H-dibenzo[a,d]cycloheptene-10-acetic acid;
(S)-10,11-Dihydro-3-[3-[4-(2-propyloxy)pyridin-2-ylamino]-1-propyloxy]-5H-dibenzo[a,d]cycloheptene-10-acetic acid;
(S)-10,11-Dihydro-3-[2-[6-(methylamino)pyridin-2-yl]-1-ethoxy]-5H-dibenzo[a,d]cycloheptene-10-acetic acid;
(S)-10,11-Dihydro-3-[3-[4-(dimethylamino)pyridin-2-ylamino]-1-propyloxy]-5H-dibenzo[a,d]cycloheptene-10-acetic acid;
(xc2x1)-10,11-Dihydro-3-[3-[4-(ethylthio)pyridin-2-ylamino]-1-propyloxy]-5H-dibenzo[a,d]cycloheptene-10-acetic acid;
(S)-10,11-Dihydro-3-[3-(4-chloropyridin-2-ylamino)-1-propyloxy]-5H-dibenzo[a,d]cycloheptene-10-acetic acid;
(xc2x1)-10,11-Dihydro-2-methyl-3-[3-(pyridin-2-ylamino)-1-propyloxy]-5H-dibenzo[a,d]cycloheptene-10-acetic acid;
(S)-10,11-Dihydro-3-[3-(4-aminopyridin-2-ylamino)-1-propyloxy]-5H-dibenzo[a,d]cycloheptene-10-acetic acid;
(xc2x1)-10,11-Dihydro-3-[3-(4-methylpyridin-2-ylamino)-1-propyloxy]-dibenzo[b,f]oxepine-10-acetic acid;
(xc2x1)-10,11-Dihydro-3-[2-[6-(methylamino)pyridin-2-yl]-1-ethoxy]-dibenzo[b,f]oxepine-10-acetic acid; and
(S)-10,11-Dihydro-3-[3-(2-aminopyridin-4-yl)-1-propyloxy]-5H-dibenzo[a,d]cycloheptene-10-acetic acid;
or a pharmaceutically acceptable salt thereof.
In cases wherein the compounds of this invention may have one or more chiral centers, unless specified, this invention includes each unique nonracemic compound which may be synthesized and resolved by conventional techniques. According to the present invention, the (S) configuration of the formula (I) compounds is preferred.
In cases in which compounds have unsaturated carbonxe2x80x94carbon double bonds, both the cis (Z) and trans (E) isomers are within the scope of this invention. The meaning of any substituent at any one occurrence is independent of its meaning, or any other substituent""s meaning, at any other occurrence.
Also included in this invention are prodrugs of the compounds of this invention. Prodrugs are considered to be any covalently bonded carriers which release the active parent drug according to formula (I) in vivo. Thus, in another aspect of this invention are novel prodrugs, which are also intermediates in the preparation of formula (I) compounds, of formula (II): 
or a pharmaceutically acceptable salt thereof.
In yet another aspect of this invention are novel intermediates of formula (III): 
or a pharmaceutically acceptable salt thereof.
Abbreviations and symbols commonly used in the peptide and chemical arts are used herein to describe the compounds of this invention. In general, the amino acid abbreviations follow the IUPAC-IUB Joint Commission on Biochemical Nomenclature as described in Eur. J. Riochem., 158, 9 (1984).
C1-4alkyl as applied herein means an optionally substituted alkyl group of 1 to 4 carbon atoms, and includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and t-butyl. C1-6alkyl additionally includes pentyl, n-pentyl, isopentyl, neopentyl and hexyl and the simple aliphatic isomers thereof. C0-4alkyl and C0-6alkyl additionally indicates that no alkyl group need be present (e.g., that a covalent bond is present).
Any C1-4alkyl or C1-6alkyl may be optionally substituted with the group Rx, which may be on any carbon atom that results in a stable structure and is available by conventional synthetic techniques. Suitable groups for Rx are C1-4alkyl, ORxe2x80x3, SRxe2x80x3, C1-4alkylsulfonyl, C1-4alkylsulfoxyl, xe2x80x94CN, N(Rxe2x80x3)2, CH2N(Rxe2x80x3)2, xe2x80x94NO2, xe2x80x94CF3, xe2x80x94CO2Rxe2x80x3, xe2x80x94CON(Rxe2x80x3)2, xe2x80x94CORxe2x80x3, xe2x80x94NRxe2x80x3C(O)Rxe2x80x3, F, Cl, Br, I, or CF3S(O)rxe2x80x94, wherein r is 0, 1 or 2.
Halogen or halo means F, Cl, Br, and I.
Ar, or aryl, as applied herein, means phenyl or naphthyl, or phenyl or naphthyl substituted by one to three substituents, such as those defined above for alkyl, especially C1-4alkyl, C1-4alkoxy, C1-4alkthio, CF3, NH2, OH, F, Cl, Br or I.
Certain radical groups are abbreviated herein t-Bu refers to the tertiary butyl radical, Boc refers to the t-butyloxycarbonyl radical, Frnoc refers to the fluorenylmethoxycarbonyl radical, Ph refers to the phenyl radical, Cbz refers to the benzyloxycarbonyl radical, Bn refers to the benzyi radical, Me refers to methyl, Et refers to ethyl. Ac refers to acetyl, Alk refers to C1-4alkyl, Nph refers to 1- or 2-naphthyl and cHex refers to cyclohexyl. Tet refers to 5-tetrazolyl.
Certain reagents are abbreviated herein. DCC refers to dicyclohexylcarbodiimide, DMAP refers to dimethylaminopyridine, DIEA refers to dilsopropylethyl amine, EDC refers to 1-(3-dimethylaminopropyl)-3-ethylcarboduimide, hydrochloride. HOBt refers to 1-hydroxybenzotriazole, THF refers to tetrahydrofuran, DIEA refers to diisopropylethylamine, DEAD refers to diethyl azodicarboxylate, PPh3 refers to triphenylphosphine, DIAD refers to diisopropyl azodicarboxylate, DME refers to dimethoxyethane, DMF refers to dimethylformamide, NBS refers to N-bromosuccinimide, Pd/C refers to a palladium on carbon catalyst, PPA refers to polyphosphoric acid, DPPA refers to diphenylphosphoryl azide, BOP refers to benzotriazol-1-yloxy-tris(dimethyl-amino)phosphonium hexafluorophosphate, HF refers to hydrofluoric acid, TEA refers to triethylamine, TFA refers to trifluoroacetic acid, PCC refers to pyridinium chlorochromate.
The compounds of formula (I) are generally prepared by reacting a compound of formula (IV) with a compound of formula (V): 
wherein R1, R2, Y and A are as defined in formula (I), with any reactive functional groups protected, and L1 is OH or halo;
and thereafter removing any protecting groups, and optionally forming a pharmaceutically acceptable salt.
Suitably, certain compounds of formula (I) are prepared by reacting a compound of formula (IV) with a compound of formula (VI): 
wherein R1, R2, Rxe2x80x2, Rxe2x80x3 and A are as defined in formula (I), with any reactive functional groups protected;
and thereafter removing any protecting groups, and optionally forming a pharmaceutically acceptable salt.
Suitably, the reaction between a compound of formula (IV) with a compound of formula (VI) is carried out in the presence of diethyl azodicarboxylate and triphenylphosphine in an aprotic solvent.
Additionally, certain compounds of formula (I) are prepared by reacting a compound of formula (IV) with a compound of formula (VII): 
wherein R1, R2, Rxe2x80x3 and A are as defined in formula (I), with any reactive functional groups protected;
and thereafter removing any protecting groups, and optionally forming a pharmaceutically acceptable salt.
Suitably, the reaction between a compound of formula (IV) with a compound of formula (VII) is carried out in the presence of diethyl azodicarboxylate and triphenylphosphine in an aprotic solvent.
Compounds of the formula (I) are prepared by the methods described in Bondinell et al., PCT Publication No. WO 97/01540 (International Application No. PCT/US96/11108), published Jan. 16, 1997, the entire disclosure of which is incorporated herein by reference.
Additionally, compounds of formula (I) are prepared by methods analogous to those described in the schemes that are detailed hereinafter. 
Scheme I details the preparation of an intermediate useful in the preparation of formula (I) compounds. 
Scheme II also details the preparation of an intermediate useful in the preparation of formula (I) compounds. 
Scheme III details the preparation of a formula (I) compound. Reaction of III-1 (which is a Scheme I-3 compound) in an aldol-type reaction with the enolate of ethyl acetate, which can be generated from ethyl acetate on exposure to an appropriate amide base, for instance lithium diusopropylamide (LDA) or lithium bis(trimethylsilyl)amide (LiHMDS), gives III-2. Frequently, THF is the solvent of choice for an aldol reaction, although THF in the presence of various additives, for instance HMPA or TMEDA, is often used. Reduction of III-2 to give III-3 (which is a Scheme II-6 compound) can be accomplished by hydrogenolysis over an appropriate catalyst, for example palladium metal on activated carbon (Pd/C), in an appropriate solvent, such as acetic acid, in the presence of a mineral acid such as HCl. Alternatively, this reduction can be accomplished by treatment of III-2 with triethylsilane in the presence of boron trifluoride etherate by the general method of Orfanopoulos and Smonou (Synth. Commun. 1988, 833). Removal of the methyl ether of III-3 to give III-4 can be accomplished with BBr3 in an inert solvent, for example CH2Cl2, or by reaction with ethanethiol and AlCl3 in an inert solvent, preferably CH2Cl2. Other useful methods for removal of a methyl ether are described in Greene, xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d (published by John Wiley and Sons). Compound 4 of Scheme 3 (III-4) is reacted with 2-[(3-hydroxy-1-propyl)amino]-4-nitropyridine-N-oxide in a Mitsunobu-type coupling reaction (Organic Reactions 1992, 42, 335-656; Synthesis 1981, 1-28) to afford III-5. The reaction is mediated by the complex formed between diethyl azodicarboxylate and triphenylphosphine, and is conducted in an aprotic solvent, for instance THF, CH2Cl2, or DMF. Compound III-5 is reacted with an alkali metal salt of an appropriate alcohol to afford III-6. Suitable alkali metals include lithium, sodium, potassium, and cesium, and the alcohol used for the displacement reaction is generally used as the solvent. Methods for forming the alkali metal salts of alcohols are well-known to those of skill in the art. The pyridine-N-oxide moiety of III-6 is reduced to the corresponding pyridine III-7 under transfer hydrogenation conditions using a palladium catalyst, preferably palladium metal on activated carbon, in an inert solvent, for instance methanol, ethanol, or 2-propanol. Cyclohexene, 1,4-cyclohexadiene, formic acid, and salts of formic acid, such as potassium formate or ammonium formate, are commonly used as the hydrogen transfer reagent in this type of reaction. The ethyl ester of III-7 is hydrolyzed using aqueous base, for example, LiOH in aqueous THF or NaOH in aqueous methanol or ethanol, and the intermediate carboxylate salt is acidified with a suitable acid, for instance TFA or HCl, to afford the carboxylic acid III-8. Alternatively, the intermediate carboxylate salt can be isolated, if desired, or a carboxylate salt of the free carboxylic acid can be prepared by methods well-known to those of skill in the art. 
Scheme IV describes an alternative method for the preparation of formula (I) compounds. Compound IV-1 is reacted with a base, preferably an alkali metal hydride such as sodium hydride or potassium hydride, in a polar, aprotic solvent, generally THF, DMF, DMSO, or mixtures thereof, to afford the corresponding alkali metal phenoxide. Alternatively, an alkali metal amide, for instance LDA, or the lithium, sodium, or potassium salt of hexamethyldisilazane, can be used for deprotonation. The intermediate phenoxide is generally not isolated, but is reacted in situ with an appropriate electrophile, for instance 2-[N-(3-methanesulfonyioxyl-propyl)-N-(tert-butoxycarbonyl)-amino]pyridine-N-oxide, to afford the coupled product IV-2. The tert-butoxycarbonyl protecting group in IV-2 is removed under acidic conditions, such as 4 M HCl in 1,4-dioxane or TFA in CH2Cl2, to afford IV-3. Conditions for removal of the tert-butoxycarbonyl protecting group are well-known to those of skill in the art, and several useful methods are described in standard reference volumes such as Greene xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d. IV-3 is subsequently converted to IV-4 following the procedure outlined in Scheme III. 
Commercially available 2-fluoro4-methoxyacetophenone (V-1) reacts with an alcohol, for example phenol, in the presence of copper metal and a suitable base, for instance K2CO3, to afford the diaryl ether V-2. On treatment with sulfur and an appropriate primary or secondary amine, preferably morpholine, according to the general method of Harris (J. Med. Chem. 1982, 25, 855), V-2 is converted to V-3 in a classical Willgerodt-Kindler reaction. The thioamide thus obtained is hydrolyzed to the corresponding carboxylic acid V-4 by reaction with an alkali metal hydroxide, suitably KOH, in an aqueous alcoholic solvent, such as aqueous MeOH, EtOH, or i-PrOH. Carboxylic acid V-4 is converted to the corresponding acid chloride by reaction with either SOCl2 or oxalyl chloride according to conditions well-known to those of skill in the art. Treatment of this acid chloride with an appropriate Friedel-Crafts catalyst such as AlCl3 or SnCl4, in an inert solvent, such as CH2Cl2 or CS2, provides the cyclic ketone V-5. Alternatively, acid V-4 can be converted directly to ketone V-5 under acidic conditions, for example with polyphosphoric acid. Reaction of V-5 in an aldol-type reaction with the enolate of ethyl acetate, which can be generated from ethyl acetate on exposure to an appropriate amide base, for instance lithium diisopropylamide (LDA) or lithium bis(trimethylsilyl)amide (LiHMDS), glues V-6. Frequently, THF is the solvent of choice for an aldol reaction, although THF in the presence of various additives, for instance HMPA or TMEDA is often used. Reduction of V-6 to give V-7 can be accomplished by treatment of V-6 with triethylsilane in the presence of boron trifluoride etherate by the general method of Orphanopoulos and Smonu (Synth. Commun. 1988, 833). Any olefinic by-products that result from elimination of the alcohol are reduced by hydrogenation over an appropriate catalyst, for example palladium metal on activated carbon (Pd/C), in an appropriate solvent, such as MeOH or EtOH. Alternatively, the reduction of V-6 to give V-7 can be accomplished by hydrogenolysis in the presence of a mineral acid such as HCl. Typically, this reaction is catalyzed by Pd/C, and is optimally conducted in acetic acid. Removal of the methyl ether of V-7 to give V-8 can be accomplished with BBr3 in an inert solvent, for example CH2Cl2, or by reaction with ethanethiol and AlCl3 in an inert solvent, preferably CH2Cl2. Other useful methods for removal of a methyl ether are described in Greene, xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d (published by John Wiley and Sons). V-8 is subsequently converted to formula (I) compounds following the procedure outlined in Scheme III.
Acid addition salts of the compounds are prepared in a standard manner in a suitable solvent from the parent compound and an excess of an acid, such as hydrochloric, hydrobromic, hydrofluoric, sulfuric, phosphoric, acetic, trifluoroacetic, maleic, succinic or methanesulfonic. Certain of the compounds form inner salts or zwitterions which may be acceptable. Cationic salts are prepared by treating the parent compound with an excess of an alkaline reagent, such as a hydroxide, carbonate or alkoxide, containing the appropriate cation; or with an appropriate organic amine. Cations such as Li+, Na+, K+, Ca++, Mg++ and NH4+ are specific examples of cations present in pharmaceutically acceptable salts.
This invention also provides a pharmaceutical composition which comprises a compound according to formula (I) and a pharmaceutically acceptable carrier. Accordingly, the compounds of formula (I) may be used in the manufacture of a medicament. Pharmaceutical compositions of the compounds of formula (I) prepared as hereinbefore described may be formulated as solutions or Iyophilized powders for parenteral administration. Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use. The liquid formulation may be a buffered, isotonic, aqueous solution. Examples of suitable diluents are normal isotonic saline solution, standard 5% dextrose in water or buffered sodium or ammonium acetate solution. Such formulation is especially suitable for parenteral administration, but may also be used for oral administration or contained in a metered dose inhaler or nebulizer for insulation. It may be desirable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate.
Alternately, these compounds may be encapsulated, tableted or prepared in a emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. Liquid carriers include syrup, peanut oil, olive oil, saline and water. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax. The amount of solid carrier varies but, preferably, will be between about 20 mg to about 1 g per dosage unit. The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulating, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.
For rectal administration, the compounds of this invention may also be combined with excipients such as cocoa butter, glycerin, gelatin or polyethylene glycols and molded into a suppository.
The compounds described herein are antagonists of the vitronectin receptor, and are useful for treating diseases wherein the underlying pathology is attributable to ligand or cell which interacts with the vitronectin receptor. For instance, these compounds are useful for the treatment of diseases wherein loss of the bone matrix creates pathology. Thus, the instant compounds are useful for the treatment of ostoeporosis, hyperparathyroidism. Paget""s disease, hypercalcemia of malignancy, osteolytic lesions produced by bone metastasis, bone loss due to immobilization or sex hormone deficiency. The compounds of this invention are also believed to have utility as antitumor, anti-angiogenic, antiinflammatory and anti-metastatic agents, and be useful in the treatment of atherosclerosis and restenosis.
The compound is administered either orally or parenterally to the patient, in a manner such that the concentration of drug is sufficient to inhibit bone resorption or other such indication. The pharmaceutical composition containing the compound is administered at an oral dose of between about 0.1 to about 50 mg/kg in a manner consistent with the condition of the patient. Preferably the oral dose would be about 0.5 to about 20 mg/kg. For acute therapy, parenteral administration is preferred. An intravenous infusion of the peptide in 5% dextrose in water or normal saline, or a similar formulation with suitable excipients, is most effective, although an intramuscular bolus injection is also useful. Typically, the parenteral dose will be about 0.01 to about 100 mg/kg; preferably between 0.1 and 20 mg/kg. The compounds are administered one to four times daily at a level to achieve a total daily dose of about 0.4 to about 400 mg/kg/day. The precise level and method by which the compounds are administered is readily determined by one routinely skilled in the art by comparing the blood level of the agent to the concentration required to have a therapeutic effect.
This invention further provides a method for treating osteoporosis or inhibiting bone loss which comprises administering stepwise or in physical combination a compound of formula (I) and other inhibitors of bone resorption such as bisphosphonates (i.e., allendronate), hormone replacement therapy, anti-estrogens, or calcitonin. In addition, this invention provides a method of treatment using a compound of this invention and an anabolic agent, such as the bone morphogenic protein, iproflavone, useful in the prevention of bone loss and/or to increase bone mass.
Additionally, this invention provides a method of inhibiting tumor growth which comprises administering stepwise or in physical combination a compound of formula (I) and an antineoplastic agent. Compounds of the camptothecin analog class, such as topotecan, irinotecan and 9-aminocamptothecin, and platinum coordination complexes, such as cisplatin, ormaplatin and tetraplatin, are well known groups of antineoplastic agents. Compounds of the camptothecin analog class are described in U.S. Pat. Nos. 5,004,758, 4,604,463, 4,473,692, 4,545,880 4,342,776, 4,513,138, 4,399,276, EP Patent Application Publication Nos. 0 418 099 and 0 088 642, Wani, et al., J. Med. Chem., 1986, 29, 2358, Wani, et al., J. Med. Chem., 1980, 23, 554, Wani, et al., J. Med. Chem., 1987, 30, 1774, and Nitta, et al., Proc. 14th International Congr. Chemotherapy., 1985, Anticancer Section 1, 28, the entire disclosure of each which is hereby incorporated by reference. The platinum coordination complex, cisplatin, is available under the name Platinol(copyright) from Bristol Myers-Squibb Corporation. Useful formulations for cisplatin are described in U.S. Pat. Nos. 5,562,925 and 4,310,515, the entire disclosure of each which is hereby incorporated by reference.
In the method of inhibiting tumor growth which comprises administering stepwise or in physical combination a compound of formula (I) and an antineoplastic agent, the platinum coordination compound, for example cisplatin, can be administered using slow intravenous infusion. The preferred carrier is a dextrose/saline solution containing mannitol. The dose schedule of the platinum coordination compound may be on the basis of from about 1 to about 500 mg per square meter (mg/m2) of body surface area per course of treatment. Infusions of the platinum coordiation compound may be given one to two times weekly, and the weekly treatments may be repeated several times. Using a compound of the camptothecin analog class in a parenteral administration, the course of therapy generally employed is from about 0.1 to about 300.0 mg/m2 of body surface area per day for about five consecutive days. Most preferably, the course of therapy employed for topotecan is from about 1.0 to about 2.0 mg/nm2 of body surface area per day for about five consecutive days. Preferably, the course of therapy is repeated at least once at about a seven day to about a twenty-eight day interval.
The pharmaceutical composition may be formulated with both the compound of formula (I) and the antineoplastic agent in the same container, but formualtion in different containers is preferred. When both agents are provided in solution form, they can be contained in an infusion/injection system for simultaneous administration or in a tandem arrangement.
For convenient administration of the compound of formula (I) and the antineoplastic agent at the same or different times, a kit is prepared, comprising, in a single container, such as a box, carton or other container, individual bottles, bags, vials or other containers each having an effective amount of the compound of formula (I) for parenteral administration, as described above, and an effective amount of the antineoplastic agent for parenteral administration, as described above. Such kit can comprise, for example, both pharmaceutical agents in separate containers or the same container, optionally as lyophilized plugs, and containers of solutions for reconstitution. A variation of this is to include the solution for reconstitution and the lyophilized plug in two chambers of a single container, which can be caused to admix prior to use. With such an arrangement, the antineoplastic agent and the compound of this invention may be packaged separately, as in two containers, or lyophilized together as a powder and provided in a single container.
When both agents are provided in solution form, they can be contained in an infusion/injection system for simultaneous administration or in a tandem arrangement. For example, the compound of formula (I) may be in an i.v. injectable form, or infusion bag linked in series, via tubing, to the antineoplastic agent in a second infusion bag. Using such a system, a patient can receive an initial bolus-type injection or infusion of the compound of formula (I) followed by an infusion of the antineoplastic agent.
The compounds may be tested in one of several biological assays to determine the concentration of compound which is required to have a given pharmacological effect.
Inhibition of Vitronectin Binding
Solid-Phase [3H]-SKandF-107260 Binding to xcex1vxcex23: Human placenta or human platelet xcex1vxcex23 (0.1-0.3 mg/mL) in buffer T (containing 2 mM CaCl2 and 1% octyiglucoside) was diluted with buffer T containing 1 mM CaCl2, 1 mM MgCl2, 1 mM MgCl2 (buffer A) and 0.05% NaN3, and then immediately added to 96-well ELISA plates (Corning, New York, N.Y.) at 0.1 mL per well. 0.1-0.2 xcexcg of xcex1vxcex23 was added per well. The plates were incubated overnight at 4xc2x0 C. At the time of the experiment, the wells were washed once with buffer A and were incubated with 0.1 mL of 3.5% bovine serum albumin in the same buffer for 1 hr at room temperature. Following incubation the wells were aspirated completely and washed twice with 0.2 mL buffer A.
Compounds were dissolved in 100% DMSO to give a 2 mM stock solution, which was diluted with binding buffer (15 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM CaCl2, 1 mM MnCl2, 1 mM MgCl2) to a final compound concentration of 100 xcexcM. This solution is then diluted to the required final compound concentration. Various concentrations of unlabeled antagonists (0.001-100 xcexcM) were added to the wells in triplicates, followed by the addition of 5.0 nM of [3H]-SKandF-107260 (65-86 Ci/mmol).
The plates were incubated for 1 hr at room temperature. Following incubation the wells were aspirated completely and washed once with 0.2 mL of ice cold buffer A in a well-to-well fashion. The receptors were solubilized with 0.1 mL of 1% SDS and the bound [3H]-SKandF-107260 was determined by liquid scintillation counting with the addition of 3 mL Ready Safe in a Beckman LS Liquid Scintillation Counter, with 40% efficiency. Nonspecific binding of [3H]-SKandF-107260 was determined in the presence of 2 xcexcM SKandF-107260 and was consistently less than 1% of total radioligand input. The IC50 (concentration of the antagonist to inhibit 50% binding of [3H]-SKandF-107260) was determined by a nonlinear, least squares curve-fitting routine, which was modified from the LUNDON-2 program. The Ki (dissociation constant of the antagonist) was calculated according to the equation: Ki=IC50/(1+L/Kd), where L and Kd were the concentration and the dissociation constant of [3H]-SKandF-107260, respectively.
Compounds of the present invention inhibit vitronectin binding to SKandF 107260 in the concentration range of about 2.5 to about 0.001 micomolar.
Compounds of this invention are also tested for in vitro and in vivo bone resorption in assays standard in the art for evaluating inhibition of bone formation, such as the pit formation assay disclosed in EP 528 587, which may also be performed using human osteoclasts in place of rat osteoclasts, and the ovarectomized rat model, described by Wronski et al., Cells and Materials 1991, Sup. 1, 69-74.
Vascular Smooth Muscle Cell Migration Assay
Rat or human aortic smooth muscle cells were used. The cell migration was monitored in a Transwell cell culture chamber by using a polycarbonate membrane with pores of 8 um (Costar). The lower surface of the filter was coated with vitronectin. Cells were suspended in DMEM supplemented with 0.2% bovine serum albumin at a concentration of 2.5-5.0xc3x97106 cells/mL, and were pretreated with test compound at various concentrations for 20 min at 20xc2x0 C. The solvent alone was used as control. 0.2 mL of the cell suspension was placed in the upper compartment of the chamber. The lower compartment contained 0.6 mL of DMEM supplemented with 0.2% bovine serum albumin. Incubation was carried out at 37xc2x0 C. in an atmosphere of 95% air/5% CO2 for 24 hr. After incubation, the non-migrated cells on the upper surface of the filter were removed by gentle scraping. The filter was then fixed in methanol and stained with 10% Giemsa stain. Migration was measured either by a) counting the number of cells that had migrated to the lower surface of the filter or by b) extracting the stained cells with 10% acetic acid followed by determining the absorbance at 600 nM.
Thyroparathyroidectomized Rat Model
Each experimental group consists of 5-6 adult male Sprague-Dawley rats (250-400 g body weight). The rats are thyroparathyroidectomized (by the vendor, Taconic Farms) 7 days prior to use. All rats receive a replacement dose of thyroxine every 3 days. On receipt of the rats, circulating ionized calcium levels are measured in whole blood immediately after it has been withdrawn by tail venipuncture into heparinized tubes. Rats are included if the ionized Ca level (measured with a Ciba-Corning model 634 calcium pH analyzer) is  less than 1.2 mM/L. Each rat is fitted with an indwelling venous and arterial catheter for the delivery of test material and for blood sampling respectively. The rats are then put on a diet of calcium-free chow and deionized water. Baseline Ca levels are measured and each rat is administered either control vehicle or human parathyroid hormone 1-34 peptide (hPTH1-34, dose 1.25 ug/kg/h in saline/0.1% bovine serum albumin, Bachem, Calif.) or a mixture of hPTH1-34 and test material, by continuous intravenous infusion via the venous catheter using an external syringe pump. The calcemic response of each rat is measured at two-hourly intervals during the infusion period of 6-8 hours.
Human Osteoclast Resorption and Adhesion Assays
Pit resorption and adhesion assays have been developed and standardized using normal human osteoclasts derived from osteoclastoma tissue. Assay 1 was developed for the measurement of osteoclast pit volumes by laser confocal microscopy. Assay 2 was developed as a higher throughput screen in which collagen fragments (released during resorption) are measured by competitve ELISA.
Assay 1 (Using Laser Confocal Microscopy)
Aliquots of human osteoclastoma-derived cell suspensions are removed from liquid nitrogen strorage, warmed rapidly at 37xc2x0 C. and washed xc3x971 in RPMI-1640 medium by centrifugation (1000 rpm, 5 mins at 4xc2x0 C.).
The medium is aspirated and replaced with murine anti-HLA-DR antibody then diluted 1:3 in RPMI-1640 medium. The suspension is incubated for 30 mins on ice and mixed frequently.
The cells are washed xc3x972 with cold RPMI-1640 followed by centrifugation (1000 rpm, 5 mins at 4xc2x0 C.) and the cells are then transferred to a sterile 15 ml centrifuge tube. The number of mononuclear cells are enumerated in an improved Neubauer counting chamber.
Sufficient magnetic beads (5/mononuclear cell), coated with goat anti-mouse IgG (Dynal, Great Neck, N.Y.) are removed from their stock bottle and placed into 5 ml of fresh medium (this washes away the toxic azide preservative). The medium is removed by immobilizing the beads on a magnet and is replaced with fresh medium.
The beads are mixed with the cells and the suspension is incubated for 30 mins on ice. The suspension is mixed frequently.
The bead-coated cells are immobilized on a magnet and the remaining cells (osteoclast-rich fraction) are decanted into a sterile 50 ml centrifuge tube.
Fresh medium is added to the bead-coated cells to dislodge any trapped osteoclasts. This wash process is repeated xc3x9710. The bead-coated cells are discarded.
The viable osteoclasts are enumerated in a counting chamber, using fluorescein diacetate to label live cells. A large-bore disposable plastic pasteur pipet is used to add the sample to the chamber.
The osteoclasts are pelleted by centrifugation and the density adjusted to the appropriate number in EMEM medium (the number of osteoclasts is variable from tumor to tumor), supplemented with 10% fetal calf serum and 1.7 g/liter of sodium bicarbonate.
3 ml aliquots of the cell suspension (per compound treatment) are decanted into 15 ml centrifuge tubes. The cells are pelleted by centrifugation.
To each tube, 3 ml of the appropriate compound treatment are added (diluted to 50 uM in the EMEM medium). Also included are appropriate vehicle controls, a positive control (anti-vitronectin receptor murine monoclonal antibody [87MEM1] diluted to 100 ug/ml) and an isotype control (IgG2d diluted to 100 ug/ml). The samples are incubated at 37xc2x0 C. for 30 mins.
0.5 ml aliquots of the cells are seeded onto sterile dentine slices in a 48-well plate and incubated at 37xc2x0 C. for 2 hours. Each treatment is screened in quadruplicate.
The slices are washed in six changes of warm PBS (10 ml/well in a 6-well plate) and then placed into fresh medium containing the compound treatment or control samples. The samples are incubated at 37xc2x0 C. for 48 hours.
Tartrate Resistant Acid Phosphatase (TRAP) Procedure (Selective Stain for Cells of the Osteoclast Lineage)
The bone slices containing the attached osteoclasts are washed in phosphate buffered saline and fixed in 2% gluteraldehyde (in 0.2M sodium cacodylate) for 5 mins.
They are then washed in water and are incubated for 4 minutes in TRAP buffer at 37xc2x0 C. (0.5 mg/ml naphthol AS-BI phosphate dissolved in N,N-dimethylformamide and mixed with 0.25 M citrate buffer (pH 4.5), containing 10 mM sodium tartrate.
Following a wash in cold water the slices are immersed in cold acetate buffer (0.1 M, pH 6.2) containing 1 mg/ml fast red garnet and incubated at 4xc2x0 C. for 4 minutes.
Excess buffer is aspirated, and the slices are air dried following a wash in water.
The TRAP positive osteoclasts (brick red/purple precipitate) are enumerated by bright-field microscopy and are then removed from the surface of the dentine by sonication.
Pit volumes are determined using the Nikon/Lasertec ILM21W confocal microscope.
Assay 2 (Using an ELISA Readout)
The human osteoclasts are enriched and prepared for compound screening as described in the initial 9 steps of Assay 1. For clarity, these steps are repeated hereinbelow.
Aliquots of human osteoclastoma-derived cell suspensions are removed from liquid nitrogen strorage, warmed rapidly at 37xc2x0 C. and washed xc3x971 in RPMI-1640 medium by centrifugation (1000 rpm, 5 mins at 4xc2x0 C.).
The medium is aspirated and replaced with murine anti-HLA-DR antibody then diluted 1:3 in RPMI-1640 medium. The suspension is incubated for 30 mins on ice and mixed frequently.
The cells are washed xc3x972 with cold RPMI-1640 followed by centrifugation (1000 rpm, 5 mins at 4xc2x0 C.) and the cells are then transferred to a sterile 15 ml centrifuge tube. The number of mononuclear cells are enumerated in an improved Neubauer counting chamber.
Sufficient magnetic beads (5/mononuclear cell), coated with goat anti-mouse IgG (Dynal, Great Neck, N.Y.) are removed from their stock bottle and placed into 5 ml of fresh medium (this washes away the toxic azide preservative). The medium is removed by immobilizing the beads on a magnet and is replaced with fresh medium.
The beads are mixed with the cells and the suspension is incubated for 30 mins on ice. The suspension is mixed frequently.
The bead-coated cells are immobilized on a magnet and the remaining cells (osteoclast-rich fraction) are decanted into a sterile 50 ml centrifuge tube.
Fresh medium is added to the bead-coated cells to dislodge any trapped osteoclasts. This wash process is repeated xc3x9710. The bead-coated cells are discarded.
The viable osteoclasts are enumerated in a counting chamber, using fluorescein diacetate to label live cells. A large-bore disposable plastic pasteur pipet is used to add the sample to the chamber.
The osteoclasts are pelleted by centrifugation and the density adjusted to the appropriate number in EMEM medium (the number of osteoclasts is variable from tumor to tumor), supplemented with 10% fetal calf serum and 1.7 g/liter of sodium bicarbonate.
In contrast to the method desribed above in Assay 1, the compounds are screened at 4 doses to obtain an IC50, as outlined below:
The osteoclast preparations are preincubated for 30 minutes at 37xc2x0 C. with test compound (4 doses) or controls.
They are then seeded onto bovine cortical bone slices in wells of a 48-well tissue culture plate and are incubated for a further 2 hours at 37xc2x0 C.
The bone slices are washed in six changes of warm phosphate buffered saline (PBS), to remove non-adherent cells, and are then returned to wells of a 48 well plate containing fresh compound or controls.
The tissue culture plate is then incubated for 48 hours at 37xc2x0 C.
The supernatants from each well are aspirated into individual tubes and are screened in a competitive ELISA that detects the c-telopeptide of type I collagen which is released during the resorption process. This is a commercially available ELISA (Osteometer, Denmark) that contains a rabbit antibody that specifically reacts with an 8-amino acid sequence (Glu-Lys-Ala-His-Asp-Gly-Gly-Arg) that is present in the carboxy-terminal telopeptide of the a1-chain of type I collagen. The results are expressed as % inhibition of resorption compared to a vehicle control,
Human Osteoclast Adhesion Assay
The human osteoclasts are enriched and prepared for compound screening as described above in the inital 9 steps of Assay 1. For clarity, these steps are repeated hereinbelow.
Aliquots of human osteoclastoma-derived cell suspensions are removed from liquid nitrogen strorage, warmed rapidly at 37xc2x0 C. and washed xc3x971 in RPMI-1640 medium by centrifugation (1000 rpm, 5 mins at 4xc2x0 C.).
The medium is aspirated and replaced with murine anti-HLA-DR antibody then diluted 1:3 in RPMI-1640 medium. The suspension is incubated for 30 mins on ice and mixed frequently.
The cells are washed xc3x972 with cold RPMI-1640 followed by centrifugation (1000 rpm, 5 mins at 4xc2x0 C.) and the cells are then transferred to a sterile 15 ml centrifuge tube. The number of mononuclear cells are enumerated in an improved Neubauer counting chamber.
Sufficient magnetic beads (5/mononuclear cell), coated with goat anti-mouse IgG (Dynal, Great Neck, N.Y.) are removed from their stock bottle and placed into 5 ml of fresh medium (this washes away the toxic azide preservative). The medium is removed by immobilizing the beads on a magnet and is replaced with fresh medium.
The beads are mixed with the cells and the suspension is incubated for 30 mins on ice. The suspension is mixed frequently.
The bead-coated cells are immobilized on a magnet and the remaining cells (osteoclast-rich fraction) are decanted into a sterile 50 ml centrifuge tube.
Fresh medium is added to the bead-coated cells to dislodge any trapped osteoclasts. This wash process is repeated xc3x9710. The bead-coated cells are discarded.
The viable osteoclasts are enumerated in a counting chamber, using fluorescein diacetate to label live cells. A large-bore disposable plastic pasteur pipet is used to add the sample to the chamber.
The osteoclasts are pelleted by centrifugation and the density adjusted to the appropriate number in EMEM medium (the number of osteociasts is variable from tumor to tumor), supplemented with 10% fetal calf serum and 1.7 g/liter of sodium bicarbonate.
Osteoclastoma-derived osteoclasts are preincubated with compound (4 doses) or controls at 37xc2x0 C. for 30 minutes.
The cells are then seeded onto osteopontin-coated slides (human or rat osteopontin, 2.5 ug/ml) and incubated for 2 hours at 37xc2x0 C.
Non adherent cells are removed by washing the slides vigorously in phosphate buffered saline and the cells remaining on the slides are fixed in acetone.
The osteoclasts are stained for tartrate-resistant acid phosphatase (TRAP), a selective marker for cells of this phenotype (see steps 15-17), and are enumerated by light microscopy. The results are expressed as % inhibition of adhesion compared to a vehicle control.
Cell Adhesion Assay
Cells and Cell Culture
Human embryonic kidney cells (HEK293 cells) were obtained from ATCC (Catalog No. CRL 1573). Cells were grown in Earl""s minimal essential medium (EMEM) medium containing Earl""s salts, 10% fetal bovine serum, 1% glutamine and 1% Penicillin-Steptomycin.
Constructs and Transfections
A 3.2 kb EcoRI-KpnI fragment of the xcex1v subunit and a 2.4 kb XbaI-XhoI fragment of the xcex23 subunit were inserted into the EcoRI-EcoRV cloning sites of the pCDN vector (Aiyar et al., 1994) which contains a CMV promoter and a G418 selectable marker by blunt end ligation. For stable expression, 80xc3x97106 HEK 293 cells were electrotransformed with xcex1v+xcex23 constructs (20 xcexcg DNA of each subunit) using a Gene Pulser (Hensley et al., 1994) and plated in 100 mm plates (5xc3x97105 cells/plate). After 48 hr, the growth medium was supplemented with 450 xcexcg/mL Geneticin (G418 Sulfate, GIBCO-BRL, Bethesda, Md.). The cells were maintained in selection medium until the colonies were large enough to be assayed.
Immunocytochemical Analysis of Transfected Cells
To determine whether the HEK 293 transfectants expressed the vitronectin receptor, the cells were immobilized on glass microscope slides by centrifugation, fixed in acetone for 2 min at room temperature and air dried. Specific reactivity with 23C6, a monoclonal antibody specific for the xcex1vxcex23 complex was demonstrated using a standard indirect immunofluorescence method.
Cell Adhesion Studies
Corning 96-well ELISA plates were precoated overnight at 4xc2x0 C. with 0.1 mL of human vitronectin (0.2 xcexcg/mL in RPMI medium). At the time of the experiment, the plates were washed once with RPMI medium and blocked with 3.5% BSA in RPMI medium for 1 hr at room temperature. Transfected 293 cells were resuspended in RPMI medium, supplemented with 20 mM Hepes, pH 7.4 and 0.1% BSA at a density of 0.5xc3x97106 cells/mL. 0.1 mL of cell suspension was added to each well and incubated for 1 hr at 37xc2x0 C. in the presence or absence of various xcex1vxcex23 antagonists. Following incubation, 0.025 mL of a 10% formaldehyde solution, pH 7.4. was added and the cells were fixed at room temperature for 10 min. The plates were washed 3 times with 0.2 mL of RPMI medium and the adherent cells were stained with 0.1 mL of 0.5% toluidine blue for 20 min at room temperature. Excess stain was removed by extensive washing with deionized water. The toluidine blue incorporated into cells was eluted by the addition of 0.1 mL of 50% ethanol containing 50 mM HCl. Cell adhesion was quantitated at an optical density of 600 nm on a microtiter plate reader (Titertek Multiskan MC, Sterling, Va.).
Solid-phase xcex1vxcex25 Binding Assay:
The vitronectin receptor xcex1vxcex25 was purified from human placenta. Receptor preparation was diluted with 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM CaCl2, 1 mM MnCl2, 1 mM MgCl2 (buffer A) and was immediately added to 96-well ELISA plates at 0.1 ml per well. 0.1-0.2 xcexcg of xcex1vxcex23 was added per well. The plates were incubated overnight at 4xc2x0 C. At the time of the experiment, the wells were washed once with buffer A and were incubated with 0.1 ml of 3.5% bovine serum albumin in the same buffer for 1 hr at room temperature. Following incubation the wells were aspirated completely and washed twice with 0.2 ml buffer A.
In a [3H]-SKandF-107260 competition assay, various concentrations of unlabeled antagonists (0.001-100 xcexcM) were added to the wells, followed by the addition of 5.0 nM of [3H]-SKandF-107260. The plates were incubated for 1 hr at room temperature. Following incubation the wells were aspirated completely and washed once with 0.2 ml of ice cold buffer A in a well-to-well fashion The receptors were solubilized with 0.1 ml of 1% SDS and the bound [3H]-SKandF-107260 was determined by liquid scintillation counting with the addition of 3 ml Ready Safe in a Beckman LS 6800 Liquid Scintillation Counter, with 40% efficiency. Nonspecific binding of [3H]-SKandF-107260 was determined in the presence of 2 xcexcM SKandF-107260 and was consistently less than 1% of total radioligand input. The IC50 (concentration of the antagonist to inhibit 50% binding of [3H]-SKandF-107260) was determined by a nonlinear, least squares curve-fitting routine, which was modified from the LUNDON-2 program. The Ki (dissociation constant of the antagonist) was calculated according to Cheng and Prusoff equation: Ki=IC50/(1+L/Kd), where L and Kd were the concentration and the dissociation constant of [3H]-SKandF-107260, respectively.
Purification of GPIIb-IIIa
Ten units of outdated, washed human platelets (obtained from Red Cross) were lyzed by gentle stirring in 3% octylglucoside, 20 mM Tris-HCl, pH 7.4, 140 mM NaCl, 2 mM CaCl2 at 4xc2x0 C. for 2 h. The lysate was centrifuged at 100,000 g for 1 h. The supernatant obtained was applied to a 5 mL lentil lectin sepharose 4B column (E.Y. Labs) preequilibrated with 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM CaCl2, 1% octylglucoside (buffer A). After 2 h incubation, the column was washed with 50 mL cold buffer A. The lectin-retained GPIIb-IIIa was eluted with buffer A containing 10% dextrose. All procedures were performed at 4xc2x0 C. The GPIIb-IIIa obtained was  greater than 95% pure as shown by SDS polyacrylamide gel electrophoresis.
Incorporation of GPIIb-IIIa in Liposomes.
A mixture of phosphatidylserine (70%) and phosphatidylcholine (30%) (Avanti Polar Lipids) were dried to the walls of a glass tube under a stream of nitrogen. Purified GPIIb-IIIa was diluted to a final concentration of 0.5 mg/mL and mixed with the phospholipids in a protein:phospholipid ratio of 1:3 (w:w). The mixture was resuspended and sonicated in a bath sonicator for 5 min. The mixture was then dialyzed overnight using 12,000-14,000 molecular weight cutoff dialysis tubing against a 1000-fold excess of 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM CaCl2 (with 2 changes). The GPIb-IIIa-containing liposomes wee centrifuged at 12,000 g for 15 min and resuspended in the dialysis buffer at a final protein concentration of approximately 1 mg/mL. The liposomes were stored at xe2x88x9270xc2x0 C. until needed.
Competitive Binding to GPIIb-IIIa
The binding to the fibrinogen receptor (GPIIb-IIIa) was assayed by an indirect competitive binding method using [3H]-SKandF-107260 as an RGD-type ligand. The binding assay was performed in a 96-well filtration plate assembly (Millipore Corporation, Bedford, Mass.) using 0.22 um hydrophilic durapore membranes. The wells were precoated with 0.2 mL of 10 xcexcg/mL polylysine (Sigma Chemical Co., St. Louis, Mo.) at room temperature for 1 h to block nonspecific binding. Various concentrations of unlabeled benzazepines were added to the wells in quadruplicate. [3H]-SKandF-107260 was applied to each well at a final concentration of 4.5 nM, followed by the addition of 1 xcexcg of the purified platelet GPIIb-IIIa-containing liposomes. The mixtures were incubated for 1 h at room temperature. The GPIIb-IIIa-bound [3H]-SKandF-107260 was seperated from the unbound by filtration using a Millipore filtration manifold, followed by washing with ice-cold buffer (2 times, each 0.2 mL). Bound radioactivity remaining on the filters was counted in 1.5 mL Ready Solve (Beckman Instruments, Fullerton. Calif.) in a Beckman Liquid Scintillation Counter (Model LS6800), with 40% efficiency. Nonspecific binding was determined in the presence of 2 xcexcM unlabeled SKandF-107260 and was consistently less than 0.14% of the total radioactivity added to the samples. All data points are the mean of quadruplicate determinations.
Competition binding data were analyzed by a nonlinear least-squares curve fitting procedure. This method provides the IC50 of the antagonists (concentration of the antagonist which inhibits specific binding of [3H]-SKandF-107260 by 50% at equilibrium). The IC50 is related to the equilibrium dissociation constant (Ki) of the antagonist based on the Cheng and Prusoff equation: Ki=IC50/(1+L/Kd), where L is the concentration of [3H]-SKandF-107260 used in the competitive binding assay (4.5 nM), and Kd is the dissociation, constant of [3H]-SKandF-107260 which is 4.5 nM as determined by Scatchard analysis.
Preferred compounds of this invention have an affinity for the vitronectin receptor relative to the fibrinogen receptor of greater than 10:1. Most preferred compounds have a ratio of activity of greater than 100:1.
The efficacy of the compounds of formula (I) alone or in combination with an antineoplastic agent may be determined using several transplantable mouse tumor models. See U.S. Pat. Nos. 5,004,758 and 5,633,016 for details of these models
The examples which follow are intended in no way to limit the scope of this invention, but are provided to illustrate how to make and use the compounds of this invention. Many other embodiments will be readily apparent to those skilled in the art.